1. Field of the Invention
The present invention pertains to the field of nucleic acid (especially DNA) ligand-based diagnostics and prophylaxis or passive “immunity” (i.e., binding and blocking infectious agents from infecting or progressing throughout the body). In particular, the application relates to single-stranded deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) ligand sequences, whether individual or linked together to form longer multiple binding site “receptors,” that specifically target and bind to arthropod-borne bacteria and viruses (arboviruses). Such arthropod-borne bacteria include the Rickettsia genus that can cause typhus or spotted fevers and deadly hemorrhagic fevers or other lethal diseases such as Crimean-Congo Hemorrhagic Fever (“CCHF”) viruses, Chikungunya (“CHIK”) viruses, Dengue viruses, and West Nile Viruses (“WNV”) or parasites such as various species of Leishmania . The arthropod vectors can include mosquitoes, ticks, lice, mites, midges, fleas, flies and sandflies.
These individual or linked DNA ligand (aptamer) sequences represent valuable target analyte-responsive components of diagnostic devices or biosensors. A biosensor can be defined as any device that employs a biologically-derived molecule as the sensing component and transduces a target analyte binding event into a detectable physical signal (including, but not limited to, changes in light intensity, absorbance, emission, wavelength, color, electrical conduction, electrical resistance, or other electrical properties, etc). Once bonded with the target, these DNA ligand sequences can be used to qualitatively determine the presence of target analyte, as well as to quantify the target analyte amount, in a sample using a broad variety of assay types and diagnostic or sensor platforms including, but not limited to: affinity-based lateral flow test strips, enzyme-linked (“ELISA-like”) microplate assays, membrane blotting, surface plasmon resonance (“SPR”), surface acoustic wave (“SAW”) or surface transverse wave (“STW”) sensors, magnetic bead (“MB”)-based capture, plastic-adherent sandwich assays, electrochemiluminescence (“ECL”), radioisotopic, fluorescence intensity assays including quantum dot (“QD”) or other fluorescent nanoparticle (“NP”)-based assays, fluorescence lifetime, and fluorescence polarization (“FP”) assays.
The invention includes general DNA ligand or aptamer-based methods of detection and quantification of these arthropod-borne diseases or related pathogens in homogenized or chemically (chaotrope or detergent)-extracted arthropods or animal or human body fluids such as whole blood, plasma, serum, sputum or saliva, interstitial, synovial, or cerebrospinal fluid aspirates, mucus, and urine or solid biopsy samples.
In addition, these DNA ligand sequences are valuable in competitive displacement assays which are not solely dependent on affinity or avidity to produce sensitive detection. Such assays would include competitive displacement fluorescence or Förster resonance energy transfer (“FRET”) assays or DNA ligand “beacon” FRET assays. Each of these types of assays and detection platforms has different applications in either central laboratories or as a component of portable detectors to identify infected arthropods (homogenates or extracts) or human or animal body fluids.
It has been established that aptamers can replace antibodies in lateral flow or chromatographic test strip assay formats and may enhance detection sensitivity by virtue of higher affinity versus comparable antibodies. Such test strips or dipsticks represent rapid, inexpensive and convenient visual detection formats. The user can add various human body fluids or arthropod homogenates or extracts (proteins removed from arthropod guts by low levels of detergents or chaotropes including guanidinium or metal salts) and obtain a positive or negative result by visualizing a red colloidal gold-aptamer conjugate line. Use of fluorescent nanoparticle (“FNP”)- or quantum dot (“QD”)-DNA aptamer conjugates on the test line of a lateral flow test strip in combination with a handheld UV penlight or common laser pointer to illuminate the fluorescent test and control lines appears to confer even greater sensitivity to the assay.
These individual or linked DNA ligand (concatamer-like aptamer) sequences represent valuable target analyte-responsive components of diagnostic devices or “biosensors.” A biosensor is defined as any sensor device that employs a biologically-derived molecule as the sensing component and transduces a target analyte binding event into a detectable physical signal, including, but not limited to, changes in light intensity, absorbance, transmittance, refraction (Surface Plasmon Resonance or SPR), wavelength, color, agglutination of cells or particles, fluorescence intensity, fluorescence lifetime, fluorescence polarization or anisotropy, fluorescence correlation spectroscopy (“FCS”), fluorescence or Förster resonance energy transfer (FRET; nonradiative dipole-dipole coupling of fluorophores or fluorophores and quenchers), upconverting phosphor (anti-Stokes shifts), two-photon interaction phenomena, Raman spectroscopy or surface-enhanced Raman spectroscopy (“SERS”), electrical conduction, electrical resistance or other electrical properties, mass, photon or radioactive particle emissions, etc.
Once bonded with the target, these DNA ligand sequences can be used to qualitatively determine the presence of analyte, as well as to quantify or semi-quantify the target analyte amount in a sample using a broad variety of assay types and diagnostic or sensor platforms including, but not limited to, affinity-based lateral flow test strips, membrane blotting, surface plasmon resonance (“SPR”), surface acoustic waveguides (“SAW”) or surface transverse waveguides (“STW”) devices, magnetic bead (“MB”)-based capture, plastic-adherent sandwich assays (“PASA”), chemiluminescence (“CL”), electrochemiluminescence (“ECL”), radioisotopic, fluorescence intensity, including quantum dot (“QD”) or other fluorescent nanoparticle (“FNP”) of dye-based, fluorescence lifetime, and fluorescence polarization (“FP”) assays, or enzyme-linked (“ELISA-like”) microplate assays.
Finally, since envelope- or capsid-protruding spike proteins on viral surfaces control binding to and invasion of host cells, the DNA ligands may have prophylactic or therapeutic value by simply binding or coating the viral spike proteins to prevent attachment to host cell surfaces and inhibiting virus entry into host cells. The prophylactic effect has been demonstrated for H5N1 influenza virus with similar DNA ligand or aptamer sequences that coated the H5N1 viruses and prevented or severely inhibited invasion of host cells and slowed or stopped subsequent viral replication.
2. Background Information
The DNA ligand sequences listed herein were derived by iterative cycles of affinity-based selection of DNA ligands from a randomized library using rickettsial or leishmanial surface molecules (cold 1.5M MgCl2-extracted outer membrane proteins; OMPs), recombinant surface proteins or synthetic peptide epitopes derived from the known amino acid sequences of viral envelope protein spikes or other surface epitopes as defined in Table 1. After affinity-based selection, the DNA ligands were subjected to polymerase chain reaction (“PCR”) amplification followed by cloning and traditional Sanger dideoxynucleotide DNA sequencing. The utility of many of the sequences in ELISA-like plate assays as well as fluorescence (intensity) assays have been used and verified as illustrated by Tables 2-7 and FIGS. 2-7 and FIG. 9.
Some of the sequences function more effectively in affinity-based (ELISA-like, lateral flow strips, or fluorescence intensity) assays, while other DNA ligand sequences against the same pathogen targets have functioned better in competitive FRET assays). Therefore, all of the listed sequences have potential utility in some assay format for use in one or more tests or types of sensors for arthropod-borne pathogens and their therapy or prevention.
Arthropod-borne pathogens can present serious threats to human health in the form of alphaviruses or flaviviruses (arboviruses) that can cause encephalitis or hemorrhagic fevers or shock syndromes and death. Similarly, untreated rickettsial infections can lead to serious cases of spotted fevers or typhus with significant mortality. Finally, visceral and non-visceral leishmaniasis are serious conditions which are difficult to treat and can be fatal. All of these diseases are transferred to man by arthropod vectors (flying or other insects including mosquitoes, fleas, mites, midges, ticks, lice, flies and sandflies).
Rapid, accurate, and ultrasensitive detection of arthropod-borne diseases aids physicians by supplying key diagnostic information in the early phases of infection. This in turn allows administration of the proper antibiotic or anti-viral agent to treat these potentially deadly diseases before they become life-threatening. Current methods of detection such as lateral flow immunochromatographic test strips, although rapid, are not very sensitive and miss early stage disease detection or rely on detection of antibodies against the disease agent which may take weeks to emerge in the patient's serum. The same is true for many slower and more tedious ELISA tests which are somewhat more sensitive than lateral flow test strips, but often rely on detection of antibodies slowly made by the patient against the infectious pathogen over a period of weeks to months. In addition, there are no truly effective therapies for some of the arboviruses.
The DNA ligands disclosed herein can potentially and directly detect many arthropod-borne pathogenic microbes with greater sensitivity and speed than conventional antibodies. These same DNA ligands or aptamers may also have value as high affinity and highly specific binding agents against arboviruses, rickettsia and parasites to block or slow disease progression.